It is commonly, but wrongly, assumed that we understand how iron gets into the brain. The problem with the existing paradigm is that it holds that the endothelial cells forming the blood-brain barrier serve simply as a conduit through which iron bound to transferrin passes. This paradigm is significantly flawed in multiple points but most notably it fails to account for the iron requirements of the endothelial cells or for a mechanism for regulating brain iron uptake. The conceptual framework for this proposal is that the endothelial cells of the BBB, far from serving as a simple conduit, are the focal point for the regulation for cerebral iron metabolism. We propose both in vivo and cell culture approaches to investigate the dynamics of brain iron uptake. We postulate that BBB endothelial cells store iron that can be released into the brain in response to external factors that are found in the extracellular fluid;thus for the first time demonstrating a regulatory system for movement of iron into the brain. Thus, upon completion of the proposed studies we will establish the significant and paradigm shifting concept that the endothelial cells control access of iron to the brain in response to their extracellular environment. In addition, this proposal will make the following specific new contributions to the existing paradigm for brain iron transport (i) add a novel mechanism for iron transport (ii) demonstrate non-transferrin-bound iron release from endothelial cells via ferroportin, a membrane-bound iron export protein (iii) reveal regulation of iron release from endothelial cells (iv) reveal endothelial cells store iron, which can be released in response to levels of proteins in the CSF or extracellular fluid (v) reveal that regulation of transferrin receptors in endothelial cells are responsive to the intracellular iron pool indicating that the iron content of the endothelial cells forming the BBB is the site at which transferrin mediated brain iron uptake is regulated. Moreover, the proposed studies will expand our knowledge of the dynamics of the iron management protein profiles in the microvasculature which will inform studies on human microvasculature such as our recent findings in the microvasculature of individuals with Restless Legs syndrome or the long-term changes in CSF protein profiles associated with iron deficiency despite the resolution of hematologic parameters. Thus, the studies proposed herein are essential to inform us about the functional significance of the altered profile status in the microvasculature and CSF in neurological disease. Most importantly, the studies are designed to understand the regulation of brain iron uptake and the adaptability of the systems for release and transport. The existence of an adaptive system in endothelial cells that responds to brain-derived signals is a completely novel concept for brain iron transport and will impact treatment concepts for developmental iron deficiency and adult neurological disorders involving impaired brain iron homeostasis. PUBLIC HEALTH RELEVANCE: The long-term goal of this line of research is to understand the dynamics of brain iron acquisition and to apply this knowledge to the maladaptions that may contribute to neurological disease. Many common neurological disorders such as Alzheimer's Disease, Parkinson's Disease and Restless Legs Syndrome involve loss of brain iron homeostasis. Moreover, iron deficiency and its associated long term cognitive and motor impairments is the most prevalent nutritional disorder in the world. Therefore understanding the mechanisms by which iron gets into the brain and how those mechanisms are regulated will position us to address significant global health issues.